This invention relates to methods and compositions for the selective modification of nucleic acids in biological compositions.
Transmission of viral diseases (e.g., hepatitis A and B, acquired immunodeficiency syndrome (HIV), cytomegalovirus infections) by blood or blood products is a significant problem in medicine. While donor selection criteria and screening of donor blood for viral markers helps reduce the transmission of viruses to recipients, screening methods are incomplete or less than 100% sensitive, as most are directed to only a few discrete viruses. Even in such cases, their sensitivity is insufficient. In addition, other biological compositions, e.g., mammalian and hybridoma cell lines, products of cell lines, milk, colostrunm and sperm, can contain infectious viruses.
It is desirable to inactivate any virus contained in donor blood, blood products, or other biological compositions. At the same time, it is important to leave the structure and function of valuable constituents, such as red blood cells, platelets, leukocytes, and plasma biopolymers, such as proteins and polysaccharides relatively unchanged.
In addition, it is often unknown whether compositions containing blood, blood products, or products of mammalian cells contain infectious viruses. In this case it would be valuable to have compositions and methods to treat such compositions to inactivate any infectious viruses present.
Furthermore, the manufacture of maximally safe and effective killed vaccines for human or veterinary use requires methods which completely and reliably render live microorganisms, e.g., viruses and bacteria, noninfectious (xe2x80x9cinactivatedxe2x80x9d) but which have minimal effects on their immunogenicity. Methods typically used for the inactivation of viruses, such as those useful in the preparation of viral vaccines, generally alter or destroy the function and structure of cells, cell components, proteins and other antigens.
Current inactivation methods, including the use of formalin, beta-propiolactone, and ultraviolet radiation, have been developed empirically, with little basis in fundamental chemical or structural principles. For example, ethyleneimine monomers have been used to inactivate the foot-and-mouth disease virus (Russian patent no. SU 1915956). Ethyleneimine monomers have also been used to inactivate Mycoplasma and Acholeplasma (WO 92/18161) and avian infections (Romania patent no. RO 101400). Binary ethyleneimine (i.e., ethyleneimine monomer generated by a combination of two reagents) has been used for the inactivation of feline enteric coronavirus, FECV, (EP 94200383). Polyethyleneimine has been used as a plant virus control agent (JP 7882735). The foregoing methods and compounds modify microorganisms, such as viruses and bacteria, nonspecifically, and are difficult to standardize and apply reproducibly.
In addition, ignorance of which chemical alterations render the microorganism noninfectious makes the process difficult to apply reproducibly. Periodic outbreaks of disease resulting from inadequate inactivation or reversion following inactivation are the result. Major outbreaks of paralytic poliomyelitis, foot and mouth disease and Venezuelan equine encephalitis have occurred due to this problem.
In general, multiple components of the microorganism, including important surface antigenic determinants such as viral capsid proteins, are affected by currently used inactivating agents. These agents significantly modify not only nucleic acids but also other biopolymers such as proteins, carbohydrates and lipids, thereby impairing their function. Altered antigens or the inactivation of protective epitopes can lead to reduced immunogenicity and hence low potency (e.g., inactivated polio vaccine), to altered antigenicity and hence immunopotentiation of disease instead of disease prevention (e.g., respiratory syncytial virus and inactivated measles vaccines produced by formalin inactivation), or to the appearance of new antigens common to another killed vaccine prepared with the same inactivant.
For example, in the preparation of hepatitis B virus vaccine, it is common practice to heat preparations at temperatures in excess of 80xc2x0 C. and to treat with formaldehyde. These treatments not only inactivate viral infectivity, but also damage proteins and other antigens. Carrier substances added to the vaccine as stabilizers also may be unintentionally modified, producing allergic reactions, as occurs with human serum albumin in rabies vaccine inactivated with beta-propiolactone.
The problems of inactivation of viruses in biological mixtures are distinct from the problems of inactivation of the viruses alone due to the co-presence of desirable biopolymers such as proteins, carbohydrates, and glycoproteins in the plasma. While it is possible to inactivate the hepatitis B virus by using agents such as formaldehyde or oxidizing agents, these methods are not suitable for the inactivation of viruses in blood, due to the observation that most of these inactivating agents impair the biological activity of biopolymers in plasma or cellular components of blood. For example, the use of ultraviolet light has been shown to inactivate viruses in a platelet concentrate. However, severe platelet damage resulted from higher doses. Beta-propiolactone reacts with nucleic acid and protein at similar rates; thus, while viruses can be inactivated, more than half of the factor VIII activity of plasma is lost.
Yet another problem is that some of the viruses contaminating blood or other biological fluids are contained within the cell, either as a fully formed virus, viral genome fragments, or viral nucleic acid integrated into the host genome. For instance, the HIV virus is contained within leukocytes. It is a special concern to be able to inactivate both cell-free and cell-contained forms of virus, while retaining the structural integrity of cells.
Problems may also exist in obtaining valuable biopolymers from non-blood sources since pathogenic viruses may also contaminate such compositions.
The invention features a method of selectively modifying nucleic acid molecules in a biological composition; the method includes the step of contacting the composition with an inactivating agent having the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. Preferably, R5 is alkylene and each of R1, R2, R3, R4, R6, R7, and R8 is H or alkyl.
The invention further features a method for selectively inactivating a virus by contacting the biological composition with this inactivating agent, where the nucleic acid molecules are contained within an infectious vertebrate virus. This method may be used for both enveloped and non-enveloped viruses. The inactivated viruses can then be included in killed vaccines.
The invention also features a method for selectively modifying nucleic acids that are contained within a transforming DNA fragment, using this inactivating agent.
The invention also features a killed vaccine that includes an effective amount of inactivated vertebrate virus and a pharmaceutically acceptable carrier, where the inactivated vertebrate virus is made by a process of incubating the virus with an inactivating agent under viral inactivating conditions. The inactivating agent has the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
The inventions also features a blood-collecting device including a container for receiving blood or a blood fraction; the container includes an inactivating agent in an amount effective to inactivate viruses in the blood or fraction thereof received into the container. The inactivating agent has the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
The invention further features a method of selectively modifying nucleic acid molecules in a biological composition; the method includes contacting the composition with an inactivating agent having the formula:
xcfx89-X1xe2x80x94[R1xe2x80x94N+(R2, R3)xe2x80x94]nR4.(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4, cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. Preferably, R1 is alkylene and each of R2, R3, and R4 is H or alkyl.
The invention further features a method for selectively inactivating a virus by contacting the biological composition with this inactivating agent, where the nucleic acid molecules are contained within an infectious vertebrate virus. This method may be used for both enveloped and non-enveloped viruses. The inactivated viruses can then be included in killed vaccines.
The invention also features a method for selectively modifying nucleic acids that are contained within a transforming DNA fragment, using this inactivating agent.
The invention also features a killed vaccine including an effective amount of inactivated vertebrate virus and a pharmaceutically acceptable carrier, where the inactivated vertebrate virus is made by a process of incubating the virus with an inactivating agent under viral inactivating conditions; the inactivating agent has the formula:
xcfx89-X1xe2x80x94[R1xe2x80x94N+(R2, R3)xe2x80x94]nR4.(X2xe2x88x92)n
where X1, is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
The invention further features a blood-collecting device including a container for receiving blood or a blood fraction; the container includes an inactivating agent in an amount effective to inactivate viruses in the blood or fraction thereof received into the container. The inactivating agent has the formula:
xcfx89-X1xe2x80x94[R1xe2x80x94N+(R2, R3)xe2x80x94]nR4.(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
The invention also features a nucleic acid inactivating agent having the formula:
xcfx89-X1xe2x80x94[R1xe2x80x94N+(R2, R3)xe2x80x94]nR4.(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. Preferably, R1 is alkylene and each of R2, R3, and R4 is H or alkyl.
The invention further features a method of selectively modifying nucleic acid molecules in a biological composition; the method includes the step of contacting the composition with an inactivating agent having the formula:
xcex2-X1xe2x80x94CH2CH2xe2x80x94N+H(R1)xe2x80x94[R2xe2x80x94N+(R3, R4)xe2x80x94]nR5.(X2xe2x88x92)n+1
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. Preferably, each of R1, R3, R4, and R5 is H or alkyl and R2 is alkylene.
The invention further features a method for selectively inactivating a virus by contacting the biological composition with this inactivating agent, where the nucleic acid molecules are contained within an infectious vertebrate virus. This method may be used for both enveloped and non-enveloped viruses. The inactivated viruses can then be included in killed vaccines.
The invention also features a method for selectively modifying nucleic acids that are contained within a transforming DNA fragment, using this inactivating agent.
The invention also features a killed vaccine containing an effective amount of inactivated vertebrate virus and a pharmaceutically acceptable carrier, where the inactivated vertebrate virus is made by a process of incubating the virus with an inactivating agent under viral inactivating conditions; the inactivating agent has the formula:
xcex2-X1xe2x80x94CH2CH2xe2x80x94N+H(R1)xe2x80x94[R2xe2x80x94N+(R3, R4)xe2x80x94]nR5.(X2xe2x88x92)n+1
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
The invention further features a blood-collecting device that includes a container for receiving blood or a blood fraction; the container includes an inactivating agent in an amount effective to inactivate viruses in the blood or fraction thereof received into the container. The inactivating agent has the formula:
xcex2-X1xe2x80x94CH2CH2xe2x80x94N+H(R1)xe2x80x94[R2xe2x80x94N+(R3, R4)xe2x80x94]nR5.(X2xe2x88x92)n+1
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
Finally, the invention features a nucleic acid inactivating agent having the formula:
xcex2-X1xe2x80x94CH2CH2xe2x80x94N+H(R1)xe2x80x94[R2xe2x80x94N+(R3, R4)xe2x80x94]nR5.(X2xe2x88x92)n+1
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. Preferably, each of R1, R3, R4, and R5 is H or alkyl and R2 is alkylene.
xe2x80x9cINACTINE(trademark)xe2x80x9d refers to compounds of the invention having (1) an aziridino moiety or a halo-hydrocarbon-amine moiety, and (2) two or more nitrogen atoms separated by hydrocarbon moieties. These compounds are also referred to as xe2x80x9cinactivating agents,xe2x80x9d or xe2x80x9cselective inactivating agents.xe2x80x9d
An inactivating agent has xe2x80x9cselectivityxe2x80x9d for nucleic acids or xe2x80x9cselectivelyxe2x80x9d reacts with nucleic acids if the comparative rate of reaction of the inactivating agent with nucleic acids is greater than the rate of reaction with other biological molecules, e.g., proteins, carbohydrates or lipids.
xe2x80x9cNucleic acidxe2x80x9d refers to both single and double stranded DNA and RNA.
xe2x80x9cBiological compositionxe2x80x9d refers to a composition containing or derived from cells or biopolymers. Cell-containing compositions include, for example, mammalian blood, red cell concentrates, platelet concentrates, leukocyte concentrates, blood cell proteins, blood plasma, platelet-rich plasma, a plasma concentrate, a precipitate from any fractionation of the plasma, a supernatant from any fractionation of the plasma, blood plasma protein fractions, purified or partially purified blood proteins or other components, serum, semen, mammalian colostrum, milk, saliva, placental extracts, a cryoprecipitate, a cryosupernatant, a cell lysate, mammalian cell culture or culture medium, products of fermentation, ascitic fluid, proteins induced in blood cells, and products produced in cell culture by normal or transformed cells (e.g., via recombinant DNA or monoclonal antibody technology). Biological compositions can be cell-free.
xe2x80x9cBiopolymerxe2x80x9d or xe2x80x9cbiological moleculexe2x80x9d refers to any class of organic molecule normally found in living organisms including, for example, nucleic acids, polypeptides, post-translationally modified proteins (e.g., glycoproteins), polysaccharides, and lipids. Biopolymer-containing compositions include, for example, blood cell proteins, blood plasma, a blood plasma fractionation precipitate, a blood plasma fractionation supernatant, cryoprecipitate, cryosupernatant or portion or derivative thereof, serum, or a non-blood product produced from normal or transformed cells (e.g., via recombinant DNA technology).
xe2x80x9cInhibit the activity of a biopolymerxe2x80x9d means to measurably decrease the function or activity of the biopolymer. The decrease in function or activity can be determined by any standard assay used to measure the activity of the particular biopolymer. For example, the inhibition of an enzyme (protein) or antigen activity can be determined by measuring changes in the rate of an enzymatic process or an immune response to the antigen using conventional assays. Another example of such inhibition is the inhibition of the genome replication, transcription, or translation of an RNA molecule that can be determined by measuring the amount of protein encoded by the RNA that is produced in a suitable in vitro or in vivo translation system.
xe2x80x9cInactivating,xe2x80x9d xe2x80x9cinactivation,xe2x80x9d or xe2x80x9cinactivate,xe2x80x9d when referring to nucleic acids, means to substantially eliminate the template activity of DNA or RNA, for example, by destroying the ability to replicate, transcribe or translate a message. For example, the inhibition of translation of an RNA molecule can be determined by measuring the amount of protein encoded by a definitive amount of RNA produced in a suitable in vitro or in vivo translation system. When referring to viruses, the term means diminishing or eliminating the number of infectious viral particles measured as a decrease in the infectious titer or number of infectious virus particles per ml. Such a decrease in infectious virus particles is determined by assays well known to a person of ordinary skill in the art.
xe2x80x9cViral inactivating conditionsxe2x80x9d refer to the conditions under which the viral particles are incubated with the selective inactivating agents of this invention, including, for example, time of treatment, pH, temperature, salt composition, and concentration of selective inactivating agent, so as to inactivate the viral genome to the desired extent. Viral inactivating conditions are selected from the conditions described below for the selective inactivation of viruses in biological compositions.
xe2x80x9cDiminish infectivity by at least 20 logs by calculationxe2x80x9d means that the decrease in the number of infectious particles is determined by calculation as described herein in Examples 5 and 6.
xe2x80x9cVirusxe2x80x9d refers to DNA and RNA viruses, viroids, and prions. Viruses include both enveloped and non-enveloped viruses, for example, poxviruses, herpes viruses, adenoviruses, papovaviruses, parvoviruses, reoviruses, orbiviruses, picornaviruses, rotaviruses, alphaviruses, rubivirues, influenza virus, type A and B, flaviviruses, coronaviruses, paramyxoviruses, morbilliviruses, pneumoviruses, rhabdoviruses, lyssaviruses, orthmyxoviruses, bunyaviruses, phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses, enteroviruses, rhinoviruses and the filoviruses.
xe2x80x9cVaccinexe2x80x9d is used in its ordinary sense to refer to an agent that is effective to confer the necessary degree of immunity on an organism while causing no morbidity or mortality. Methods of making vaccines are, of course, useful in the study of the immune system and in preventing animal or human disease.
xe2x80x9cPharmaceutically acceptablexe2x80x9d means relatively non-toxic to the animal to whom the compound is administered. xe2x80x9cPharmaceutically acceptable carrierxe2x80x9d encompasses any of the standard pharmaceutical carriers, buffers and excipients such as water, and emulsions, such as oil/water or water/oil emulsions, and various types of wetting agents and/or adjuvants.
The methods and compositions of the present inventions provide advantages over other approaches to selectively modifying nucleic acids in the presence of other biomolecules, and in the presence of cells. As the inactivating agents described herein are selective for the nucleic acids that make up viruses, viruses can be selectively inactivated over the other molecules present.
Other features and advantages of the invention will be apparent from the following description and from the claims.
The invention features methods for selectively modifying nucleic acids in a biological composition by contacting the composition with an inactivating agent. The nucleic acids in the composition are chemically modified at rates much faster than those of the other biological molecules. The methods are therefore useful in any process in which the practitioner wishes to modify nucleic acids, while leaving other biological molecules relatively unchanged. For example, the methods can be used to inactivate viruses selectively.
The inactivating agents of the present invention include both aziridino compounds and halo-hydrocarbon-amine compounds.
The aziridino compounds have the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. These compounds can be prepared by the aziridine-initiated oligomerization of a halo-hydrocarbon-amino compound.
The halo-hydrocarbon-amine compounds can have the formula xcfx89-X1xe2x80x94[R1xe2x80x94N+(R2, R3)xe2x80x94]nR4.(X2xe2x88x92)n, where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R2, R3, and R4 cannot all be H when R1 contains 2 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. These compounds can be prepared by the oligomerization of the corresponding halo-hydrocarbon-amino compounds.
Alternatively, these compounds can have the formula xcex2-X1xe2x80x94CH2CH2xe2x80x94N+H(R1)xe2x80x94[R2xe2x80x94N+(R3, R4)xe2x80x94]nR5.(X2xe2x88x92)n+1, where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety having 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive. These compounds can be prepared by the aziridine-initiated oligomerization of an halo-hydrocarbon-amino compound, followed by conversion of the aziridino group to the corresponding halide compound.
The first step in viral inactivation according to the invention involves the physical association of an inactivating agent with nucleic acids through electrostatic interactions. The inactivating agents described above have multiple positively charged atoms, and are therefore oligocations. As oligocations, they have a high affinity for oligoanions. The association constant is proportional to the oligocation and oligoanion volume charge densities and therefore increases with the total average positive charge of the oligocation. Longer oligomers will have a higher total average positive charge, leading to an increase in their association constant with polynucleotides, for example with viral RNA. As nucleic acids are the major polyanionic components of viruses, this step results in the selective binding of the inactivating agents to viral nucleic acid, rather than to other virion components.
In addition, the distances between the positively charged nitrogen atoms of the inactivating agents is similar to the distances between the intemucleotide phosphate groups (which are negatively charged) of polynucleotides.
In the case of aziridino compounds, the second step of viral inactivation is the protonation of the aziridino group. The reactivity of aziridines as electrophilic agents increases dramatically with the protonation of the aziridine nitrogen. Therefore, the form of these compounds protonated at the aziridino group is the more reactive form. The rates of usual electrophilic reactions of aziridines should be directly proportional to the concentration of their protonated forms in the complexes with polynucleotides.
The degree of protonation depends, in part, upon pH. In solution, the pK of the aziridino group decreases markedly as the total positive charge of the molecule increases. At a pH of about 7.0, the proportion of reactive aziridino groups in many inactivating agents is low. However, after association with a polyanion, the pK of the aziridino group increases significantly. Therefore, when the aziridino compound binds to a nucleic acid, the fraction of reactive aziridino groups increases locally.
The inactivating agents of this invention modify nucleic acids preferentially through the reaction of the aziridino group, or through the reaction of the terminal halo-hydrocarbon-amine group with the nucleic acid bases in polynucleotides. The action of these compounds on polynucleotides leads to amino-alkylation of nucleophilic groups in the nucleic acid bases.
Aziridines, like many electrophilic agents, modify nucleic acids preferentially at N7, N3, and N1 of purines and to a much lesser extent at N3 of pyrimidines. Template synthesis is arrested by alkylating agents primarily due to relatively slow opening of the imidazole ring of N7 alkylated purines, predominantly of guanine. For example, aziridine modifies guanosine to produce N7(aminoethyl)-guanosine which displays a much higher rate of imadozole ring opening than does N7-alkylguanosine.
In the absence of repair or recombination, the modification of the nucleotide bases blocks replication, transcription, and translation of the viral genome and renders the virus non-infectious. Virion coat proteins, however, are not modified to the same extent.
As the above discussion illustrates, it is the electrostatic interactions between the positively charged groups on the INACTINE(trademark) and the negatively charged phosphate groups on the DNA and RNA backbones that result in the selectivity of the INACTINE(trademark) for nucleic acids. The exact structures of the INACTINE(trademark) are therefore not as critical as, for example, the structures of many pharmaceutically useful compounds. For example, a change in one of the R substituents from a methyl group to an ethyl group will not significantly affect an INACTINE(trademark) compound""s ability to selectively modify nucleic acids over other biomolecules.
What is important, however, is that the reactive part of the molecule (i.e., the aziridino group or the halo-hydrocarbon-amine group) remains reactive. For example, the aziridine ring will lose some of its reactivity if it is substituted by more than two hydrocarbon groups.
In addition, if the compounds contain hydrocarbon groups that have more than 4 carbon atoms, the compounds become lipophilic. Lipophilicity is undesired, as it will cause the agents to modify compounds such as proteins.
Aromatic rings are also undesirable substituents, as aromatic rings will intercalate in the DNA or RNA. The resulting change in the DNA or RNA structure disrupts the binding of the inactivating agents of the invention with the DNA or RNA.
The rate of modification of any virion component by traditional inactivating agents is usually considered a function of the average solution concentration of the agent. If a low-molecular-mass agent has a specific affinity for some polymer, however, the local concentration of agent near this polymer is higher than the average solution concentration of the agent and exponentially decreases with increased distances from the polymer. The selectivity of viral genome inactivation should be proportional to the difference in the local concentration of the agent near these biopolymers. Therefore, even a local increase in inactivating agent concentration near the genome should preferentially increase the modification rate of the genome.
However, as considered above, the formation of complexes between inactivating agents and polynucleotides should increase the extent of aziridino group protonation, and hence, the rate constant (k1) of polynucleotide modification. Because of the exponential decrease in agent concentration with distance, at 1-2 nm away from the polymer the local concentration of agent is essentially the same as its average solution concentration. Obviously, the fraction of the reagent reactive form at this distance should be the same as in the free (non-associated) state in solution.
Therefore, the increase in the concentration of the inactivating agent in the vicinity of the polynucleotide, as well as the association of the inactivating agent with polynucleotide, should not affect the modification rate of the capsid component, especially their antigen-bearing regions at the surface of the virion, of a protein, or of other macromolecules in the surrounding solution.
The practitioner can determine the extent of alkylation of a viral nucleic acid by the extent of viral infectivity inactivation using various assays known to a person of ordinary skill in the art, such as determination of cytopathic effect (CPE) in tissue culture using serial dilutions of virus-containing mixtures introduced into susceptible cells, followed by incubation at 37xc2x0 C. Modification of proteins, polysaccharides and glycoproteins with inactivating agents would lead to the introduction of additional positive charges. The extent of this biopolymer modification can be determined by means known in the art including, for example, isoelectric focusing, polyacrylamide gel electrophoresis, HPLC, and other forms of chromatography with detection by autoradiography or a suitable method.
All these data and considerations allow one to select an inactivating agent, with the desired polynucleotide affinity, leading to an increased rate of reaction and selectivity of the viral genome modification. Thus, even if the selectivity of the aziridine or halo-hydrocarbon-amino moiety is no better than the selectivity of other agents now used for preparation of whole virion killed vaccines, the significant increase in selectivity of the oligomeric inactivating agents makes negligible the effect of virion component modification on immunogenicity, stability and other virion properties.
Viruses can be inactivated by contacting a composition containing the virus with about 0.0001 M to about 0.015 M of an inactivating agent in a solution having an ionic strength of about 0.01 M to about 0.5 M, at a pH of about 6.5 to about 7.5, at a temperature of about 4xc2x0 C. to about 45xc2x0 C. The concentration of the inactivating agent depends, in part, on the number of positively charged atoms in the molecule. The selection of pH depends, in part, on the stability of the virion. The salts used can be any of those normally used in biochemical applications, including sodium, potassium, acetate, and so on. The practitioner can adjust the pH of the solution using many buffers customarily used in the art to handle biopolymers or cells, such as acetate, HEPES, MOPS, and so forth. The practitioner can also adjust other factors such as concentration of the reactants, temperature, and time of incubation. It should be kept in mind, however, that the reaction rate is dependent upon the ionic strength of the solution.
Experimentally, a decrease in infectivity can be measured to at least about xe2x80x9c6 logsxe2x80x9d in a cell- or biopolymer-containing composition. This means that the virus is inactivated to the extent determined by infectivity studies, where that virus is present in the untreated serum in such a concentration that even after dilution to 106, viral activity can be measured. When a specific virus cannot be produced to a titer of 106, inactivation is determined by direct quantitation measured up to the titer of virus produced. Alternatively, such a decrease in the number of infectious virus particles is determined by calculation as described herein to the extent of at least about xe2x80x9c20 logsxe2x80x9d based upon a kinetic description of the inactivation process based on a precise experimental determination of the infectivity of the viral suspension during inactivation while taking into account chemical, physical and biological factors affecting inactivation kinetics.
The inactivated viruses are made by a process of treating viruses under viral inactivating conditions effective to diminish infectivity to a desired extent (to at least about 6 logs by direct measurement or to at least 20 logs by calculation as described herein).
As described above, the inactivating agents of the invention can be used to selectively inactivate viruses in biological composition. In addition, the inactivating agents of the invention can be used to selectively modify nucleic acids that are contained within a transforming DNA fragment, for example, DNA from a polyoma virus.
Killed vaccines can be made by contacting a virus with a selective inactivating agent under viral inactivating conditions. The viral inactivating conditions are selected from the methods described above for modifying viral, bacterial or other nucleic acids. In general, virus at a titer of about 107 to 108 units per ml is incubated with inactivating agent at about pH 6.5 to about pH 7.5, in a solution having an ionic strength of less than about 0.5 M at about 4xc2x0 C. to about 40xc2x0 C. The time of treatment (i.e., the end point of inactivation) depends on the structure and composition of the particular virus, temperature of incubation, ionic strength, and the number of protonizable or positively charged groups in the inactivating agents. However, kinetic studies indicate that depending on pH and the virus to be inactivated, incubation time could be as little as a few seconds, and also can be about 1 hour, 5 hours, 50 hours, 100 hours 300 hours or 500 hours. The killed virus can be used directly in vaccine formulations, or lyophilized in individual or multiple dose containers for subsequent mixture with the pharmaceutically acceptable carrier. Methods of preparing vaccines are well known in the art and can be found, for example, in Vaccines (Slorein, G. Martance, E. eds) Second edition 1994, Saunders Harcourt-Brace, Phil, Toronto.
The vaccines of this invention are useful in the prevention of animal or human disease. Vaccines capable of conferring the desired degree of immunity will, of course, contain an amount of inactivated virus effective to evoke an immune response. In the preparation of killed vaccines, the sample of virus is incubated with the selective inactivating agents of this invention in amounts and under such conditions to inactivate the virus while retaining immunogenicity.
The vaccine can be administered in an adjuvant, i.e., a substance that potentiates an immune response when used in conjunction with an antigen. The vaccine can be given in an immunization dose. An immunization dose is an amount of an antigen or immunogen needed to produce or enhance an immune response. The amount will vary with the animal and immunogen or antigen or adjuvant but will generally be less than about 1000 xcexcg per dose. The immunization dose is easily determined by methods well known to those skilled in the art, such as by conducting statistically valid host animal immunization and challenge studies. See, for example Manual of Clinical Immunology, H. R. Rose and H. Friedman, American Society for Microbiology, Washington, D.C. (1980).
Suitable pharmaceutical carriers and their formulations are described in Martin, Remington""s Pharmaceutical Sciences, 91th Ed. (Mack Publishing Co., Easton 1995). Such compositions will, in general, contain an effective amount of the compound together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the subject.
The particular dosage of the vaccine to be administered to a subject will depend on a variety of considerations including the nature of the virus, the schedule of administration, the age and physical characteristics of the subject, and so forth. Proper dosages may be established using clinical approaches familiar to the medicinal arts.
Methods of treating cell- or biopolymer-containing compositions or preparing killed vaccines are particularly useful in the inactivation of viruses already known in the art to be irreversibly inactivated by other alkylating agents, such as ethyleneimine monomer and xcex2-propiolactone. Therefore, while the agents of this invention have broader use by virtue of their selectivity, in selecting viruses for the preparation of vaccines or biological products for decontamination, the practitioner is guided, in part, by experience in the art with other inactivating agents.
The methods and compositions of the present invention can also be used to inactivate blood-transmitted viruses, bacteria, or parasites in cell- or biopolymer-containing compositions in various contexts, e.g., in the hospital, laboratory, or as part of a kit. Since cell compositions also comprise a variety of proteins, the method of viral inactivation described herein is also applicable to protein fractions, particularly blood plasma protein fractions or purified blood products. These include, but are not limited to, fractions containing clotting factors (such as factor VIII and factor IX), serum albumin and/or immune globulins. The viral and bacterial inactivation may be accomplished by treating a protein fraction or purified protein with a selective inactivating agent as described herein.
The process of the invention can be combined with still other modes of inactivating viruses. For example, certain processes used in the preparation of medical products (e.g., chromatography in buffers of low pH, or storage of red blood cells in acidic solutions containing calcium chelating agents) may have incidental viral inactivating properties for selected, sensitive viruses, usually enveloped viruses.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.